肝素寡醣體晶片的製備及其應用

Preparation of Heparin Oligosaccharide Chips for Screening Heparin-binding Proteins

潘冠榮(Guan-Rong Pan)

指導教授 ： 廖文峯 ； 洪上程

國立清華大學/理學院/化學系/博士(2010年)

摘要

Abstract

Cell-surface heparan sulfate (HS) and its structurally related heparin (HP) play significant roles in a diverse set of biological processes, including cell-cell communication, viral infection, and molecular recognition. However, these materials are not readily available from nature. In this dissertation, we aim to prepare HS/HP oligosaccharides and resolve the difficulties encountered during the synthesis. With the synthesized oligosaccharides, surface plasmon resonance technology would be used to detect the role and relationship of these sugars with other biological molecules.
Chapter 1 initially describes the major components of the cell surface carbohydrates. Structural characterizations as well as biological properties of HP/HS are then discussed to provide a deeper understanding of the relationship between HP and HS. Chapter 2 provides a brief introduction and reviews the literature on the synthesis of heparin octasaccharide or longer sugar chain in recent years. The specific aims and the retro-synthetic plans to address the new preparative routes are illustrated in Chapter 3.
Chapter 4 outlines the construction of even-numbered HP/HS oligosaccharide skeletons from nonreducing end to reducing end, and in particular, the improvements on the reaction pathway. In Chapter 5, the synthesis of odd-numbered fully-protected HP and HS skeleton is detailed. The methodology utilized a linker-attached monosacchatide which was coupled with the even-numbered skeletons to obtain the target compound.
The synthesis of HP/HS di-, tetra-, hex and octasaccharides through an eight-step functional groups transformation of the corresponding sugar skeletons is illustrated in Chapter 6. Chapter 7 presents the attachment of the prepared compounds into a chip together with the investigation of interaction with the proteins FGF1 and HBHA. Strong binding with FGF1 was detected.
The conclusion of our synthetic work is summarized in Chapter 8. Finally, Chapter 9 provides the detailed experimental procedure and the characterization of all new compounds.

摘要
硫酸乙醯肝素(heparan sulfate, HS)與肝素(heparin, HP)是屬於葡胺聚醣(glycosaminoglycan, GAG)的生物分子。到目前為止已經被發現有許多生理反應或病毒的感染皆是透過和硫酸乙醯肝素的鍵結，但是硫酸乙醯肝素在自然界中不容易取得，為研究硫酸乙醯肝素與其他生物分子(例如:病毒、細菌、或生物體重要的蛋白質)間作用的機制。本論文將著重於如何使用化學合成來製備結構確定且成份均勻的硫酸乙醯肝素的寡醣體，再利用電漿表面共振技術，探討其與蛋白質的作用關係。
這篇論文分成九個章節，第一章主要是在描述細胞表面的醣類種類，並將重點著重在描述硫酸乙醯肝素與肝素兩者的結構與生物活性。第二章則次針對近幾年來醣類生物化學家，對有關合成肝素八醣或八醣以上的報導。第三章則是提出我們想要解決的幾個問題及我們想要合成的目標分子，並試著將目標分子進行反合成分析。
第四章描述了如何由非還原端來建構偶數肝素寡醣體的骨架，並且改進合成的路徑。第五章描述了我們對於構成硫酸乙醯肝素與肝素的奇數醣體骨架的的合成工作，在遇到一些問題之後，改由具丙烯基的單醣體骨架配合連結體重新建構單醣，並成功完成骨架建立工作。第六章是描述改變連結單元接上寡醣體骨架以後，經由八個步驟就可以得到硫酸乙醯肝素的雙醣體、四醣體、六醣體以及八醣體。最後，第七章提出了製成硫酸乙醯肝素的醣晶片後，針對於蛋白質FGF1和HBHA測試，並且在與FGF1的偵測中，獲得不錯的結果。
第八章結論中，我們統整了第四到第七章的合成與測試成果。第九章則是提供了在合成工作中所需要的實驗步驟與新產生化合物的詳細物理性質與光譜資料。

關鍵字

肝素和硫酸乙醯肝素 ； 醣晶片

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